Photodynamic Therapy (hereinafter, referred to as PDT) has been widely introduced in clinical practice for the management of malignant tumors. One of the main factors specifying PDT efficiency is targetability or selectivity, which represents the extent of selective accumulation of photosensitizers only in tumor tissue, but not in healthy tissue. High targetability improves the efficiency of PDT to shorten the treatment period, and also to reduce the side effects of the drug that is introduced into the body. Activation of a photosensitizer by light at specific wavelengths leads to the production of reactive oxygen species such as singlet oxygen and radical species. The generated reactive oxygen species directly destroys tumor cells, and induces immune inflammatory responses and damage to the microvasculature of the tumor. Most photosensitizers tested accumulate with some selectivity in tumors, but they also concentrate in normal tissues, including the skin.
Targeted delivery of the photosensitizer could solve these problems through an enhanced photocytotoxicity as a result of higher and more selective accumulation in the tumor cells. Targeting implies conjugation of the photoactive compound to a tumor-seeking (specific) molecule, either directly or by the use of a carrier. Several photosensitizers have been already conjugated with antibodies directed against tumor-associated antigens. Ligands such as low-density lipoprotein, insulin, steroids, transferrin, and epidermal growth factor (EGF) have all been described for ligand-based targeting of photosensitizers to cells overexpressing the receptors for these ligands.
In fact, alterations in receptor expression, increased levels of specific cell surface membrane lipids and proteins as well as changes in the cellular microenvironment, all occur in diseased cells.
Among the different strategies for implementing receptor-mediated delivery systems, the receptor for folic acid also constitutes a useful target for tumor-specific drug delivery due to the following reasons.
(1) Folate receptors are upregulated in many human cancers, including malignancies of the ovary, colon, mammalian gland, and lung, and renal cell carcinoma, brain metastasis of epithelial tumor, and neuroendocrine carcinoma.
(2) Access to the folate receptor in normal tissues almost always does not occur since expression of the folate receptor in normal tissues can be severely limited due to its location on the apical membrane of polarized epithelia.
(3) The density of polarized epithelia and the folate receptor increases (the density of the folate receptor appears to increase as the stage/grade of the cancer worsens).
(4) Folate has a high affinity for its cell surface receptor. Conjugation of folic acid to macromolecules has been shown to enhance their delivery to folate receptor-expressing cancer cells in vitro in almost all situations tested.
Receptor of folic acid (RFA) is a glycosylphosphatidylinositol glycoprotein which binds with folic acid to facilitate folate uptake into cells via receptor-mediated endocytosis.
Although the precise mechanism of folate receptor transport of folic acid into cells via RFA remains unresolved, it is clear that folate conjugates are taken up nondestructively by mammalian cells via receptor-mediated endocytosis.
Physiologic folates move across the plasma membrane into the cytoplasm by a specialized endocytosis mediated pathway. After binding to folate receptor on the cancer cell surface, folate conjugates, regardless of size, are seen to absorb in intracellular compartments called endosomes.
Generally, the degree of selectivity or targetability does not exceed the ratio of 10:1 (cancer cell:normal cell). Accordingly, methods of selectively delivering the photosensitizer to the membrane receptor of specific cell groups by linking to the cell surface-specific vector ligand, such as antibodies, oligosaccharides, transferrin, and hormone analogs have been developed. Many studies reveal that conjugation of a chemotherapeutic agent with these vectors increases targeted delivery by 5-10 folds higher than non-conjugation. The cells are able to bind with the conjugate via receptor-mediated endocytosis without their destruction.
Folic acid consists of three components, and belongs to the class of vitamins.
Living organisms have mainly reduced into folic acid forms, such as dihydrofolic acid, tetrahydrofolic acid, and 5-methyl-tetrahydrofolic acid. They are cofactors for enzymes in which catalyze transportation of single carbon units. Folate-dependent enzymes participate in the biosynthesis of purine and pyrimidine nucleotides, and also in the metabolism of amino acids such as methionine, histidine, serine and glycine. Thus, folates are essential for cell division and growth.
After intake in vivo, folates are rapidly absorbed in blood and transported to tissues with blood plasma and erythrocytes.
Animal cells cannot synthesize folates. Hence, a specific system in the cell membrane for binding and absorption of folates is required.
As far as folic acid is a dianion with hydrophilic properties, it poorly penetrates through the cell plasma membrane by means of simple diffusion. Only at high pharmacological concentrations does passive diffusion contribute to folic acid transport.
Under natural physiological conditions, folic acid is available in the tissues and blood serum at nanomolar concentrations. That is why cells require a highly effective membrane system for absorption and transport of the vitamin.
A mobile carrier catalyzes folic acid transport at a high rate. The carrier is abundant in the epithelial cells of the small intestine, where folic acid absorption occurs. Catalyzed transport is the main route of folic acid absorption in various cells and a substrate of such transport is folic acid in restored form. For this reason, the carrier is called a transporter of restored vectors (TRV), which is a glycoprotein of 46 kD, forming a “channel” that permits hydrophilic molecules to pass through the cell membrane. The kinetics of TRV-mediated transport is described using Michaelis-Menten kinetics. The transport rate is rather high, and its affinity to folic acid is relatively low, about 200 μM.
TRV also operates in tumor cells. The affinity of restored folic acid, KM is in the range of 1-4 μM. The affinity of the carrier for methotrexate has a slightly lower KM in the range of 4-8 μM, and its maximum rate is in the range of 1-12 nmol/min per cellular protein (g). TRV functions in the folate transport across cell membranes, but its affinity for oxygenated folic acid is low (KM is in the range of 100-200 μM).
A receptor-mediated system functions through a membrane glycoprotein called a folate receptor. A folate receptor is very similar to substrata in that its association constant for folate is less than 1 nM.
The receptor-mediated transport of folic acid takes place in one direction, namely, to take folate into cells. Normal cells express very few folate receptors on their surfaces, although with some exceptions. However, high levels of folate receptor on the cell surface are observed in malignant transformed cells, in particular, tumor cells in the lung, kidney, brain, large intestine, and ovary, and myelocytic blood cells in leukemia. Due to the increase in its quantity, folate receptors are more efficiently capable of binding with a significant amount of folic acid (more than 6·107 molecules per cell). As far as it is shown that monoclonal antibodies used for cancer diagnostic purposes bind with folic acid with high specificity, such glycoprotein could be referred to as a tumor marker.
The receptor-mediated transport of folic acid is driven by endocytosis. The receptor operates through a recirculatory mechanism. That is, while a ligand repeats binding and release of molecules, the molecules are transported across the plasma membrane into endosomes, and in the opposite direction. Efficiency of this function is specified by various factors as follows: the number of receptors on the cell surface, extracellular concentration of folic acid-ligand, affinity of folate for receptor, the rate of energy-dependent endocytosis, the release rate of receptor molecules from endosome, and the capability of the receptor to be repeatedly reintegrated into the membrane, etc.
A folate receptor-associated fraction in folate-drug conjugates will traffic into the cells by receptor-mediated endocytosis, while the remainder will remain on the cell surfaces. In this regard, two types of therapeutic strategies can be envisioned. Drugs that require access to intracellular targets can be delivered in substantial quantities to cytosolic locations by the endocytic pathway, while drugs that can or must function at an extracellular location will be enriched on cancer cell surfaces by the stationary population of the folate receptor.
An important feature is the direct delivery of the drug to pathologically transformed cells. The therapeutic effect of PDT using a number of photosensitizers is mediated by changing the physiological conditions of the pathological focus, but not by direct damage to tumor cells. Thus, hydrophilic pigments, in particular, chlorin e6 cause photodamage to the vascular system of tumor tissue (the effect of PDT to blood vessel), which inhibits tumor growth without direct inactivation of the transformed cells. It is apparent that selective delivery of photosensitizers to tumors could be one of the ways to improve the therapeutic effects of PDT.
Chlorin e6 is a natural compound, and non-toxic to normal cells of organisms. It also has higher photochemical activity on malignant cells than other photoactive compounds used in tumor therapy.
Chlorin e6 drugs quickly proceed from blood and organs to tumor affected areas, and accumulate in tumor cells at high therapeutic concentrations.
Laser-activated chlorin e6 directly destroys tumor cells as well as having indirect specific anti-tumor immunomodulating effect at the cost of cell immunity. High accumulations of chlorin e6 in the inflammatory foci or regenerating tissues provides better healing of post-operative wounds and prevents reinfection.